Redox Chemistry and [Au(CN)2-] in the Formation of Gold Metabolites

The role of hypochlorite ion, which can be generated by the enzyme myleoperoxidase, in the biochemistry of gold(I) anti-arthritic drugs was investigated. Sodium hypochlorite (OCl−) directly and rapidly oxidizes AuSTm, Au(CN)2-, AuSTg (gold thioglucose) and auranofin (Et3PAuSATg). The resulting gold(III) species were detected by an Ion Chromotography Ion-Pairing technique that was developed to distinguish gold(I) and gold(III). Formation of Au(III) was also demonstrated spectrophotometrically after the conversion to AuCl4−. The reactions of AuSTm, AuSTg, and auranofin are complex and gold(III) appears only after the initial oxidation of the thiolate (and phosphine) ligands. The enzymatic reaction, using MPO with H2O2 and Cl− as substrates, leads to slow oxidation of Au(CN)2-, AuSTm or AuSTg. The extent and rate of reaction depend on the concentrations of MPO, H2O2, and Au(I). The continued presence of Au(I) during the initial stages of reaction (oxidation of the thiolates in AuSTm and AuSTg) and the conversion to Au(III) in the latter stages of the reaction were demonstrated. Au(CN)2-, a gold metabolite, binds tightly to serum albumin. Unlike other gold(I) complexes, aurocyanide reacts almost negligibly at Cys-34 via ligand exchange. Instead, there is a strong association (K1 = 5.5 × 104 and K2 = 7.0 × 103; n1 = 0.8 and n2 = 3) of intact Au(CN)2-. The full extent of binding is revealed only by equilibrium methods such as NMR or ultrafiltration; the bound gold dissociates extensively on conventional gel-exclusion columns and partially on Penefesky spun columns. The immunological and pharmacological significance of these results are discussed.


INTRODUCTION
The mechanism of action of anti-arthritic gold drugs has been a matter of speculation and research for almost six decades. Strong evidence suggests that the bulk of gold in vivo, especially the circulating metabolites, remain gold(I), the oxidation state present in myochrysin, solganol, and auranofin [1]. Thus, it is interesting that immunological results in animal models [2] and humans [3] suggest a role for gold(Ill). This prompted us to reexamine the cursory report [4] that gold(I) can be oxidized by hypochlorite ion generated from H202 and CIin the presence of the enzyme myleoperoxidase (MPO) which is produced and released by phagocytic cells.
The oxidation of gold(I) thiomalate (AuSTm) to gold(Ill) by myleoperoxidase-generated hypochlorite ion was briefly described in an abstract by Beverly and Couri [4]. Their reactions were effected under conditions mimicking the oxidative burst of phagocytic immune cells. Although further details were reported in a thesis [5], no full report has appeared in the standard literature and these studies were limited to the single gold complex, AuSTm. The protein chemistry of gold complexes has been explored in some detail [1] but relatively less is known about its biological redox chemistry. A number of lines of evidence suggest that gold(Ill) is readily reduced to gold(I) under conditions prevailing in vivo [6,7,8] and that bulk gold in tissues is gold(I) even after administration of gold(Ill) to laboratory animals [8]. Nonetheless, many reviews point out the feasibility of oxidation [9][10][11][12][13] and possible reactions have been reported [4,14,15]. Thus, the observation that gold(Ill), but not gold(I), induces secondary immune responses in laboratory animals [2] and human patients [3] treated with gold(I) drugs suggests that gold(Ill) is indeed generated in vivo.
Since generation of gold(Ill) by stimulated macrophages or polymorphoneutrophils has the potential to dramatically alter the metabolism and anti-arthritic activity, as well as the immunogenicity, of gold drugs, we have investigated the direct reaction of sodium hypochlorite with gold thiomalate, gold(I)thioglucose, aurocyanide and auranofin. The MPO-mediated oxidation of these species was also examined. To facilitate the analysis, we developed an Ion Chromotography Ion-Pairing technique to quantitate the +1 and +111 oxidation states of gold after forming dicyanogold(I) and tetracyanogold(lll), respectively, in situ.
Conditions under which the same enzyme, MPO, can produce cyanide from thiocyanate and subsequently generate aurocyanide [Au(CN)2-] from AuSTm have also been reported [16].
Therefore, it was also of interest to examine the reaction(s) of aurocyanide with serum albumin, the principle gold binding protein in blood.

EXPERIMENTAL PROCEDURES
Materials. Gold Thiomalate (H2AuSTm; aurothioapfelsiure, lot #192129) was a kind gift from E. Time/min deionized) with mM tetrabutylammonium hydroxide, 500 #M Na2CO3. Samples were introduced via a 50 #L loop and eluted at mL/min. Au(CN)2and Au(-CN),standard solutions were prepared in water and used for elution time and sensitivity calibrations.

Oxidation AuSTm by Hypochlorite
Reactions of OCIand AuSTm were initially carried out with 40 #M gold and 174 #M OCI-.
Repetitive scans at one minute intervals demonstrated the rapid conversion of AuSTm into a new species with a more intense absorbance in the 220 nm region followed by a slower change in the new absorption band. Because the spectrum of gold(Ill) in aqueous solution at neutral pH results from a mixture of hydroxo and aquo species in a complex equilibrium [21 ], the reaction mixture was subsequently acidified with HCI to a concentration of 100 mM. The resulting spectrum revealed a band at 316 nm characteristic of AuCI 4which demonstrates oxidation of the gold(I) to gold(Ill).
The absorbance corresponded to 22.2 #M Au(lll) (40% conversion) in the original solution after 40 min. No gold(Ill) was detected in control solutions containing 160 or 320 #M OCIbut no AuSTm, or 40 #M AuSTm but no OCI-.
The incomplete reaction observed above could be due to slow kinetics of oxidation, an equilibrium process, or competing oxidation of the thiomalate ligand. The first possibility was eliminated by studying extent of reaction between 40/M AuSTm and 160 or 320/M OCIover 30 minutes. As shown in Figure  but dependent on the concentration of OCI-. 320 #M hypochlorite (80CI-/Au(I)) drove the reaction to completion, but 160 #M OCI-(40CI-/Au(I)) yielded only 55% conversion. Next a titration was performed measuring the production of Au(lll) as a function of the OCIconcentration. As shown in Figure 2, the reaction consumes between 2.5 and 3 equivalents of hypochlorite before the onset of Au oxidation. This corresponds to the oxidation of the thiomalate to its sulfonic acid form, TmSO 3 before the gold can be oxidized: AuSTm + 3OCI-AuCI 2 + OCI + 3H + > AuCI 2 + TmSO 3 + CI > Au III + H20 + 3CI- The ability of the thiomalate to neutralize three equivalents of hypochlorite was confirmed by an analogous tritration of bis(thiomalato)gold(I), generated in situ from reaction of one equivalent of thiomalate with AuSTm. In this case extensive formation of gold(Ill) occurred only after five equivalents of hypochlorite were added, Fig. 2.
The presence of unoxidized gold(I) when fewer than 3 or 6 equivalents of OCIhad been added to AuSTm or Au(STm)2 respectively, was confirmed by addition of cyanide ion (to mM final concentration). Au(I) is converted to Au(CN)2 which has four unusually sharp and strong diagnostic bands in the region 200-240 nm, Fig. 3. The formation constant of Au(CN)2is unusually large [23,24]. Facile and complete displacement of thiolates from Au(I) by cyanide has been previously demonstrated [19][20][21]. Thus, the presence of Au(I) can be demonstrated qualitatively in the early but not the later stages of the titration.

Ion Chromatographic Analysis of Au(l) & Au(lll)
To eliminate the ambiguity inherent in UV-visible assessment of the presence of gold(I) and gold(Ill), a more reliable and quantitative method was developed. Ion chromotography [25] using an anion column with ion pairing technique was found to be suitable for the detection of gold(I) and gold(Ill) as their respective cyanide complexes: AuI(CN)2 and AulII(cN)4 Conductivity measurements provide a sensitive and reproducible method to quantitate the two ions as they elute from the anion column. Au(CN)2 eluted as a sharp peak at about 3.9 min and Au(CN)2as a broader peak at about 6.7 min. The limits of detection were 55 and 110 nM, respectively; and the linear ranges were found to be 3 to 45 #M and 0.5 to 6 #M. Table gives the concentrations of gold(I) and gold(Ill) in solutions of AuSTm after reaction with to 3 equivalents of OCI-. The IC data clearly confirm that the formation of gold(I) commences only after extensive oxidation of the thiomalate ligand.

Hypochlorite Oxidation of Other Gold(I) Compounds
AuSTg is also oxidized to Au(lll) by OCIin a reaction that shares the characteristics of the AuSTm reaction: preliminary oxidation of the thiolate ligand and then conversion to gold(Ill). The reaction may be slightly slower as the reaction reaches its endpoint after 2 minutes, not within the first minute as for AuSTm.
The hypochlorite oxidation of Au(CN)2 was clearly demonstrated by the loss of the characteristic UV bands at 204, 211,230 and 240 nm [30]. These results indicate that the reactions of AuSTm and AuSTg are not limited to the gold(I) thiolates but are a characteristic of gold(I) complexes in general. The presence of Au(lll) was confirmed by the absorbance of AuCI 4and by IC.
Auranofin (Et3PAuSAtg), which contains a triethylphosphine ligand as well as a thiolate (2,3,4,6-tetra-O-acetyl-l-thiolato-8-D-glucose) was also examined. The formation of Au(lll) commenced in this case after four equivalents of hypochlorite were added, which is consistent with the oxidationo.f the phosphine to Et3PO and thiolate to sulfonate preceeding the oxidation of gold(I) to gold(Ill). ]P NMR results confirm the formation of Et3PO (A.A. Isab, A. Jagarlamudi, and C.F. Shaw III, unpublished result).

Myleoperoxidase Mediated Oxidation of ,Gold
Myleoperoxidase is an important enzyme of the oxidative burst. It is present in neutrophils and to a lesser extent other phagocytic immune cells. During the oxidative burst, it utilizes hydrogen peroxide to generate hypochlorite from chloride ion: H202 + CI-> OCI-+ H20.
It is likely then that hypochlorite generated by myleoperoxidase can then effect the oxidation of gold(I) to gold(Ill) under in vivo conditions. To explore this possibility, gold(I) compounds were exposed to MPO in the presence of its substrates, H202 and chloride ion, to ascertain the feasibility and the extent of oxidation that would occur. PBS was used as the reaction medium because the degranulation of neutrophils releases MPO into the extracellular space which has a neutral pH.
The reaction of AuSTm with H20. and CIin the presence of MPO led to a complex pattern of changes in the absorbance monitored at 220 nm, Fig. 4. There was an initial decrease over about 1-2 minutes followed by an increase over the next 2-3 minutes after which the absorbance leveled off or declined very slowly. The same pattern was observed by Beverly [5].
Control reactions in which CI-, H202, AuSTm or MPO were omitted from the mixture failed to produce the characteristic decrease and increase in absorbance. Beverly [5] had hypothesized that the initial decrease is due to the initial oxidation of the thiomalate which disrupts the gold-thiolate bonds giving rise to broad absorbances in the region 200-240 nm. The inorganic studies above and IC data below provide strong support for this interpretation. The subsequent increase in absorbance at 220 nm is due to the formation of Au(lll) as a mixed chloro, aquo, hydroxo complex. Increasing the AuSTm concentration increased the time required to achieve the minima and maxima in absorbance, further indicating that both phases involve this complex.
The presence of Au(lll) in the product mixture was confirmed by UV-vis spectroscopy after conversion to AuCI 4 by adding HCI to 0.1 M and by Ion Chromatography after conversion to Au(CN)4 by adding HCN to mM. Fig. 5 shows ion chromatographs of the same reactant mixture examined in Fig. 4 before and at 1, 2, 3 and 10 min. following the initiation of reaction. The decrease in the gold(I) signal (eluting 3.9 min after injection) and the increase in the gold(Ill) signal (6.7 min after injection) are clearly delineated. The final sample taken 10 minutes after initiating the reaction shows no detectable gold(I), indicating that complete oxidation was effected.
Oxidation of Au(CN) 2and AuSTg have also been examined in the presence of MPO, H202 and CIto generate OCI-. The aurocyanide reaction proceeds with an absorbance increase at 220 nm after a brief lag phase (ca. 15 sec). The increase is completed within about 2 minutes and shows a gradual decrease of the next 5-10 minutes. The conversion to Au(lll) was confirmed by demonstrating the presence AuCI 4 after adding HCI to 0.10 M. As the AuSTg reaction proceeds, the 220 nM absorbance increases and then levels off after about 2-3 min. Thus, it spectrophotometrcally.

Au(lll) was again confirmed
Binding of Aurocyanide to Serum Albumin Au(CN)2 .can be generated in the presence of $CNunder conditions approximating those found in wvo during chrysotherapy [23] and is found in patients who are non-smokers [24].
These recent reports demonstrate that Au(CN)2is an important metabolite and that its role in gold therapy warrants investigation. Many gold complexes react with serum albumin at cys-34 to form thermodynamically robust complexes which are transported in the blood [1]. Au(CN)-, unlike AuSTm or AuSTg, accummulates rapidly and extensively in red blood cells, especially =n patients who smoke [26]. Thus, it is important and necessary to characterize the interaction(s) of aurocyanide and serum albumin in order to understand the transport and distribution of this metabolite.
Preliminary studies [Dr. A. A. Isab,27] with Au(13CN) by.C-13 NMR spectroscopy suggested extensive association with albumin that was =ndependent of Cys-34, while chromatographic studies with Au(14CN)2 suggested a very limited extent of reaction via ligand exchange at Cys-34 to to form AIbSAuCN. To resolve the inherent contradictions additional studies were undertaken using two additional separation methods. The first is Penefsky chromatography which rapidly separates free and protein-bound metal ions over a gel-exclusion column using centrifugal force [28]. The second method is membrane ultrafiltration which preserves the equilibrium between bound and free metal ion during the separation procedure [29]. aSephadex G-50 column (1 x 25 cm) eluted by gravity flow with 100 mM NH4HCO3 Buffer, pH 7.9; ,1.1 mM albumin. Sephadex G-25 column (2 ml syringe) eluted centrifugally with 10 mM phosphate buffer, pH 7.4 + 100 mM NaCI; 0.4 mM albumin. CFiltron(R) concentrators (3000 MW cutoff) centrifuged at 5000 x g for 45 min; 10 mM phosphate buffer, pH 7.4 + 100 mM NaCI; 0.4 mM albumin. Table II shows the results of the measurements. When one equivalent of Au(CN)2 was reacted with BSA, the Au/BSA ratio found after separation by conventional chromatography, Penefsky  The labile equilibrium binding of aurocyanide to albumin provides a conceptual basis for rationalizing the disparate results of the three separation methods. At equilibrium, extensive binding is observed and, as expected for the association constants, the measured Au/BSA ratio is large. The conventional columns were eluted over a period exceeding an hour. The albumin moves through the column more rapidly than the trailing low molecular weight species, allowing the Au(CN)2 to dissociate and be irreversibly lost. The Penefsky columns are rapidly eluted. During the short time that the albumin is in contact with the resin (< min) there is relatively less opportunity for dissociation so that an intermediate value is obtained.

DISCUSSION
The oxidation of gold(I) to gold(Ill) was found previously for AuSTm [4,5] and here for AuSTm, Au(STm)2-, AuSTg, Au(CN)2and auranofin to be complete with micromolar concentrations of gold andhypochlorite. The plausibility of these findings can be evaluated using standard reduction potentials [31,32] HOCl/OCl-). Graham etaL [23] have demonstrated that myleoperoxidase-generated hypochlorite can convert physiological concentrations of SCNto CNwhich in the presence of AuSTm reacts further to form Au(CN)2-. It has been shown here that Au(CN)2can be oxidized to Au(lll). If an excess of cyanide is present, Au(CN)4will form due to the thermodynamically favorable and kinetically facile ligand exchange reactions that favor cyanide over chloride, thiocyanate or water ligands.
Thus, the gold(Ill) species formed in viva may be Au(CN)4as shown in the flow diagram of The favorable thermodynamics and the rapidity of the oxidation of gold(I) by thiomalate suggest that a gold(I)-gold(lll) redox cycle can be established in viva: OCI-(MPO generated) thiols, thioethers, disulfides Our studies of the reduction of AuCI 4and Au(CN)4 by serum albumin and various thiols and thioethers (Isab, Jagaralmudi, Schraa and Shaw, unpublished) suggest that the reduction is somewhat slower than hypochlorite oxidation of gold(I). When the forward reaction occurs as rapidly as hypochlorite is generated, as found here, and the reverse reaction over a few minutes, kinetic considerations imply that a significant fraction of the gold present at the site of the oxidative burst can accumulate in the form of gold(Ill). This powerful oxidant can then diffuse away from the site and react with protein reductants that are encountered.
This, in turn, can have immunological consequences associated with both the clinical benefits and the side effects of chrysotherapy. Oxidation of proteins can convert self-proteins into "non-self proteins" causing peptides presented by HLA molecules of antigen-presenting cells to elicit immune responses as found in several side effects to gold therapy. Alternatively, this oxidation could alter the structure of proteins that are already being detected as non-self in the pathogenesis of rheumatoid arthritis. This could, in turn, inhibit their binding to HLA molecules, and, therefore, minimize or inhibit their subsequent recognition by T-cells leading to an observable clinical improvement in the disease state. Further research on the details of gold(Ill) formation and its re-reduction by model proteins and the consequences of such reactions for the generation of cryptic peptides during antigen processing are crucial to a more detailed understanding of the mechanism of action of gold-based anti-arthritic agents.